Interactions between cytoplasmic myosin and actin filaments are thought to be responsible for a variety of motile activities in nonmuscle cells. As in muscle contraction, hydrolysis of ATP by myosin provides the required energy. While the actin-activated ATPases of vertebrate smooth muscle and nonmuscle myosins are regulated by phosphorylation of their 20,000-dalton light chains, we have found that their mechanisms of activation are very different. An elctrophoretic method was developed to separate thymus myosin with two phosphorylated light chains from unphosphorylated myosin and myosin with one phosphorylated light chain. This technique was used to show that the two heads of thymus myosin are phosphorylated randomly. This apparent lack of cooperatively contrasts with the reported ordered phosphorylation of gizzard smooth muscle myosin. The actin-activated ATPase of thymus myosin was found to increase linearly with the fraction of light chain phosphorylated. Since the two heads of thymus myosin are phosphorylated randomly, this linear correlation shows that phosphorylation of one head of thymus myosin stimulates the actin-activated ATPase of that head independent of the phosphorylation of the second head. In contrast, both heads of gizzard myosin must be phosphorylated for actin to activate the ATPase of either head. Analysis of the dependence of the ATPase activity of unphosphorylated thymus myosin on actin concentration showed that phosphorylation does not alter the maximum rate of ATP hydrolysis, but rather it causes a 15-fold increase in the apparent affinity of this myosin for actin. In the presence of tropomysoin, phosphorylation cases only about a 2-fold increase in affinity of thymus myosin for actin. Phosphorylation of gizzard myosin, on the other hand, causes a large increase in the maximum rate of ATP hydrolysis and only a small change in affinity of this myosin for actin.